*Organism (including strain): Dengue Virus type 3 - Singapore/8120/1995
Viruses; ssRNA positive-strand viruses, no DNA stage; Flaviviridae; Flavivirus; Dengue virus group.
Etiologic Risk Group :
Figure 1: Distribution of dengue, Western Hemisphere
Figure 2: Distribution of dengue, Eastern Hemisphere
*Background/Disease Information :
Dengue Fever & Dengue Hemorrhagic Fever
( Also known as :Hemorrhagic dengue; Dengue shock syndrome; Philippine hemorrhagic fever; Thai hemorrhagic fever; Singapore hemorrhagic fever )
The four dengue viruses originated in monkeys and independently jumped to humans in Africa or Southeast Asia between 100 and 800 years ago. Dengue remained a relatively minor, geographically restricted disease until the middle of the 20th century.
The 4 dengue viruses (DENV) are immunologically related, positive-strand RNA viruses of the genus Flavivirus, family Flaviviridae. These viruses cause both dengue fever (DF) and dengue hemorrhagic fever (DHF). Asymptomatic infection also occurs. Infection with 1 DENV produces lifelong immunity against reinfection with that DENV type but no long-term cross-protection against the other 3 DENV types (any cross-protection is ≤2 months).
Early symptoms of dengue hemorrhagic fever are similar to those of dengue fever, but after several days the patient becomes irritable, restless, and sweaty. These symptoms are followed by a shock -like state.
Bleeding may appear as tiny spots of blood on the skin (petechiae) and larger patches of blood under the skin (ecchymoses). Minor injuries may cause bleeding.
Shock may cause death. If the patient survives, recovery begins after a one-day crisis period.
No specific therapeutic agents exist for dengue virus infections. Encourage bed rest and maintenance of fluids to prevent dehydration while the patient is febrile. Control fever with acetaminophen. Headache, eye pain, joint pain, and muscle ache may require narcotics. Aspirin, aspirin-containing drugs, and other nonsteroidal anti-inflammatory drugs (such as ibuprofen) should be avoided because of their anticoagulant properties. Aspirin and other salicylates should be especially avoided in children because of the association with Reye syndrome.
Synthetic Product
A trace of impurity might be detectable by TLC when 100 µg is applied to the plate. The amount of contamination detectable by HPLC is less than 2 %.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3U1I_C
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
*length of your protein in Amino Acids:130 amino acids (from 1344 to 1473) Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 14065.2
*GC% Content for gene: 46.7 % *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATG AAC AAT CAA AGA AAA AAG ACA GGA AAG
CCC TCA ATC AAT ATG CTC AAA AGA GTC AGG
AAC AGG GTC AGC ACA GGA AGT CAA CTC GCT
AAA AGA TTC TCT AAA GGA CTG TTG AAC GGA
CAG GGA CCA ATG AAG TTG GTG ATG GCA TTC
ATC GCC TTT CTC AGA TTC CTC GCA ATA CCA
CCT ACA GCA GGG GTC TTA GCA AGA TGG GGA
ACA TTT AAA AAG AGT GGA GCT ATA AAG GTC
CTA AAA GGG TTT AAG AAA GAA ATA TCA AAT
ATG CTG TCC ATA ATT AAT CAG AGA AAA AAG
ACT AGT CTG TGT CTC ATG ATG ATA CTG CCA
GCT GCA CTG GCT TTC CAC CTC ACC TCA AGA
GAC GGA GAG CCA AGA ATG ATC GTG GGA AAA
AAC GAG AGA GGA AAG TCC CTT CTA TTT AAG
ACC GCC TCA GGA ATA AAC ATG TGT ACA CTC
ATC GCA ATG GAT CTT GGA GAA ATG TGC GAT
GAC ACA GTT ACA TAT AAG TGT CCA CAT ATC
ACA GAG GTT GAA CCA GAG GAT ATC GAC TGT
TGG TGT AAT CTC ACT TCA ACT TGG GTG ACT
TAC GGA ACA TGC AAC CAG GCA GGG GAG CAC
AGA AGG GAC AAA AGG AGC GTG GCT CTG GCA
CCA CAC GTG GGC ATG GGA CTC GAT ACC AGG
ACA CAG ACA TGG ATG TCT GCA GAG GGA GCT
TGG AGA CAA GTT GAA AAG GTT GAA ACA TGG
GCC TTG AGA CAT CCA GGA TTT ACC ATT TTG
GCC CTT TTT CTC GCA CAT TAT ATA GGA ACA
TCA CTT ACT CAA AAG GTG GTT ATC TTT ATA
CTC TTA ATG TTG GTG ACA CCA TCA ATG ACA
ATG AGG TGC GTT GGC GTG GGA AAC AGA GAC
TTC GTG GAG GGA CTT AGC GGA GCA ACC TGG
GTG GAT GTT GTC TTG GAA CAT GGC GGA TGC
GTC ACA ACT ATG GCA AAA AAC AAA CCA ACC
CTG GAT ATA GAA CTG CAA AAA ACT GAA GCA
ACT CAG TTG GCT ACA CTA AGA AAA CTA TGC
ATA GAA GGC AAA ATC ACT AAT ATT ACA ACC
GAC TCC AGG TGT CCA ACC CAA GGA GAG GCC
GTG TTA CCA GAG GAA CAG GAT CAA AAT TAT
GTG TGT AAA CAC ACT TAT GTG GAT AGA GGC
TGG GGA AAT GGA TGC GGG CTC TTC GGG AAG
GGC TCT CTC GTC ACA TGC GCA AAG TTC CAA
TGT TTG GAA CCA ATA GAA GGA AAG GTC GTG
CAA TAC GAG AAC CTT AAA TAT ACA GTT ATA
ATT ACA GTG CAC ACA GGA GAT CAA CAC CAG
GTG GGC AAT GAG ACA CAA GGA GTG ACA GCC
GAG ATA ACA CCC CAG GCT TCA ACA ACC GAA
GCA ATA TTG CCC GAA TAC GGA ACT CTG GGA
TTG GAG TGC AGC CCC AGA ACT GGA TTG GAT
TTT AAT GAA ATG ATA TTG CTG ACA ATG AAA
AAC AAG GCC TGG ATG GTC CAC AGA CAA TGG
TTT TTC GAC CTC CCA CTG CCA TGG GCA AGC
GGG GCT ACA ACT GAG ACC CCA ACT TGG AAT
AGG AAA GAA CTC CTT GTT ACA TTC AAA AAT
GCC CAC GCA AAA AAG CAA GAA GTT GTC GTG
CTT GGA AGC CAG GAA GGA GCT ATG CAC ACA
GCT CTC ACA GGG GCT ACA GAG ATA CAA AAC
TCA GGA GGC ACT TCC ATT TTT GCA GGC CAC
TTG AAG TGC AGA TTG AAA ATG GAT AAA CTG
GAA TTA AAG GGA ATG TCA TAC GCC ATG TGT
ACA AAT ACA TTC GTG CTG AAG AAA GAG GTT
AGC GAA ACA CAG CAT GGA ACC ATC TTA ATT
AAG GTG GAA TAT AAA GGA GAA GAC GCT CCT
TGT AAA ATT CCA TTT TCC ACT GAA GAC GGA
CAA GGA AAA GCA CAT AAC GGC AGA CTC ATC
ACC GCA AAT CCC GTG GTT ACA AAA AAG GAA
GAG CCT GTG AAC ATC GAA GCC GAG CCC CCA
TTC GGG GAG AGC AAT ATC GTG ATC GGA ATA
GGA GAC AAT GCA CTA AAA ATT AAT TGG TAT
AAA AAG GGA TCA AGC ATC GGA AAG ATG TTC
GAG GCT ACT GAA AGA GGA GCC AGA AGG ATG
GCC ATT TTG GGA GAT ACA GCA TGG GAC TTC
GGG TCC GTG GGA GGC GTC TTG AAT TCA CTG
GGA AAA ATG GTG CAC CAA ATA TTT GGA TCA
GCA TAT ACT GCA TTG TTC TCA GGA GTC TCA
TGG GTG ATG AAG ATA GGA ATA GGA GTC CTC
TTG ACA TGG ATT GGA CTG AAC TCA AAA AAT
ACA AGT ATG AGT TTC TCC TGT ATA GCT ATT
GGA ATA ATC ACC TTG TAC CTG GGA GCT GTT
GTG CAG GCA GAC ATG GGA TGC GTG ATT AAC
TGG AAA GGC AAA GAA CTG AAG TGC GGA TCT
GGC ATC TTT GTC ACA AAC GAG GTC CAT ACA
TGG ACA GAA CAG TAC AAG TTT CAA GCA GAT
TCA CCA AAA AGA CTC GCT ACT GCT ATT GCT
GGA GCA TGG GAG AAC GGA GTC TGT GGA ATA
AGA AGC ACC ACT AGA ATG GAA AAT TTG TTA
TGG AAG CAA ATA GCA AAT GAA CTG AAT TAT
ATA TTG TGG GAA AAT AAC ATA AAG TTA ACA
GTT GTG GTT GGA GAC ACA CTA GGA GTC CTA
GAA CAA GGA AAA AGG ACA CTG ACA CCT CAA
CCA ATG GAG TTA AAA TAC TCT TGG AAA ACA
TGG GGC AAG GCT AAG ATA GTG ACC GCA GAG
ACA CAG AAT TCA AGC TTC ATT ATA GAC GGC
CCA AAC ACT CCA GAA TGT CCA TCA GCC TCA
AGA GCT TGG AAT GTC TGG GAA GTC GAA GAT
TAC GGG TTC GGG GTC TTT ACC ACA AAT ATC
TGG CTG AAG CTG AGA GAA GTG TAC ACA CAG
TTG TGC GAC CAT AGA TTA ATG TCC GCA GCT
GTT AAG GAC GAG AGA GCA GTT CAC GCC GAC
ATG GGA TAT TGG ATT GAA TCA CAA AAA AAC
GGC TCT TGG AAA CTA GAG AAA GCA TCA CTC
ATC GAG GTG AAA ACT TGC ACA TGG CCA AAA
TCA CAC ACA TTG TGG ACA AAT GGA GTG TTA
GAG TCA GAT ATG ATA ATT CCA AAG TCC CTG
GCT GGG CCA ATT AGT CAA CAT AAC TAT AGA
CCA GGA TAT CAC ACA CAG ACA GCT GGG CCA
TGG CAC TTG GGA AAA CTT GAG TTG GAC TTT
AAT TAT TGT GAA GGG ACC ACT GTG GTT ATT
ACA GAG AGC TGC GGA ACA AGA GGA CCC TCC
CTA AGA ACC ACA ACT GTT TCT GGG AAA TTA
ATA CAC GAA TGG TGT TGC AGA TCA TGT ACA
TTG CCC CCA TTG AGG TAT ATG GGG GAG GAT
GGA TGT TGG TAT GGA ATG GAG ATA AGA CCA
ATA TCT GAA AAG GAG GAA AAC ATG GTT AAA
TCC CTT GTG AGC GCT GGA TCA GGG AAA GTT
GAC AAC TTC ACA ATG GGG GTG TTA TGC TTG
GCT ATT CTT TTT GAG GAA GTG CTA AGA GGA
AAG TTC GGA AAA AAG CAT ATG ATT GCC GGA
GTG TTC TTT ACA TTT GTC CTA TTA CTA TCA
GGG CAA ATA ACA TGG AGA GAC ATG GCA CAT
ACA CTA ATA ATG ATT GGA TCA AAC GCC TCA
GAC AGA ATG GGG ATG GGG GTT ACA TAC CTG
GCA CTG ATT GCA ACA TTT AAA ATT CAA CCA
TTC TTG GCC TTA GGG TTC TTT CTC AGA AAA
TTG ACA TCA AGG GAA AAC CTA TTA CTG GGA
GTC GGC CTG GCA ATG GCT ACA ACC CTA CAA
CTC CCA GAA GAC ATC GAA CAG ATG GCC AAC
GGG GTG GCA CTT GGC CTG ATG GCT CTT AAA
CTT ATA ACA CAA TTC GAA ACA TAT CAG CTT
TGG ACA GCT CTG GTT TCA CTT ACA TGT AGT
AAT ACA ATC TTC ACA CTT ACA GTG GCT TGG
AGA ACA GCC ACA TTG ATT CTA GCA GGA GTT
AGC CTT TTG CCC GTC TGC CAA TCA TCT AGC
ATG AGA AAG ACT GAT TGG TTG CCT ATG ACA
GTG GCT GCC ATG GGA GTG CCT CCA CTG CCT
CTT TTT ATT TTC TCA TTA AAA GAC ACA TTG
AAA AGA AGG TCA TGG CCA TTG AAC GAG GGG
GTT ATG GCA GTC GGA CTC GTG AGT ATC CTA
GCA TCA TCT TTA CTT AGA AAT GAC GTC CCA
ATG GCA GGA CCA TTG GTG GCC GGG GGA CTG
CTT ATT GCC TGC TAC GTC ATA ACA GGA ACT
TCA GCT GAT CTG ACT GTT GAA AAG GCA CCA
GAT GTG ACT TGG GAA GAG GAA GCC GAG CAA
ACA GGA GTC AGC CAT AAC CTA ATG ATA ACT
GTT GAC GAT GAC GGC ACA ATG AGA ATA AAG
GAT GAC GAA ACA GAA AAC ATA CTG ACA GTG
TTG CTG AAA ACA GCA TTG TTA ATC GTT TCT
GGC ATA TTC CCC TAT AGC ATT CCC GCC ACC
TTG TTA GTT TGG CAC ACA TGG CAA AAA CAA
ACC CAA AGA TCT GGA GTG CTG TGG GAC GTG
CCT TCA CCT CCC GAA ACA CAG AAA GCT GAG
CTA GAA GAG GGA GTC TAC AGA ATC AAA CAA
CAG GGG ATA TTT GGG AAA ACA CAA GTC GGC
GTG GGA GTG CAA AAG GAA GGC GTG TTT CAC
ACA ATG TGG CAC GTC ACA AGA GGG GCT GTC
TTG ACA CAT AAC GGA AAG AGA CTG GAG CCT
AAT TGG GCT AGT GTC AAG AAA GAT CTC ATC
AGC TAT GGA GGC GGG TGG AGG TTG TCA GCA
CAG TGG CAG AAG GGA GAG GAA GTG CAA GTG
ATA GCC GTG GAA CCC GGC AAA AAT CCA AAA
AAC TTT CAA ACC ACA CCC GGG ACC TTC CAA
ACA ACT ACA GGA GAA ATT GGG GCA ATA GCC
TTA GAC TTC AAA CCA GGG ACT TCT GGA TCA
CCC ATT ATC AAT AGG GAG GGA AAG GTG GTT
GGA CTG TAC GGC AAC GGA GTG GTT ACA AAG
AAC GGA GGG TAC GTG AGT GGA ATT GCA CAG
ACA AAT GCT GAA CCC GAT GGA CCA ACA CCA
GAA TTG GAG GAA GAG ATG TTT AAA AAG AGA
AAT TTG ACA ATA ATG GAT CTT CAC CCC GGA
TCA GGA AAG ACA AGG AAA TAC CTA CCA GCT
ATA GTG AGA GAA GCT ATA AAA AGG AGA TTG
AGG ACC TTG ATA CTA GCA CCA ACA AGA GTG
GTC GCT GCA GAG ATG GAG GAA GCA CTC AAA
GGA CTC CCT ATC AGG TAC CAG ACA ACT GCC
ACC AAA AGC GAA CAT ACT GGG AGA GAA ATC
GTC GAC TTG ATG TGC CAT GCC ACA TTC ACA
ATG AGA CTC CTA AGC CCC GTG AGG GTG CCA
AAC TAC AAC TTG ATT ATC ATG GAT GAG GCA
CAC TTT ACT GAC CCT GCC TCT ATT GCA GCT
AGA GGG TAT ATA AGC ACA AGA GTG GGG ATG
GGG GAA GCA GCC GCA ATA TTT ATG ACA GCC
ACA CCA CCT GGA ACT GCT GAT GCA TTC CCT
CAG TCA AAC GCC CCA ATA CAG GAT GAG GAA
AGA GAT ATC CCA GAA AGA TCA TGG AAC TCA
GGA AAT GAA TGG ATT ACA GAC TTC GCT GGC
AAG ACA GTG TGG TTT GTG CCA TCA ATT AAG
GCT GGC AAT GAC ATC GCC AAC TGT CTT AGA
AAG AAC GGA AAA AAG GTG ATA CAG CTA TCA
AGG AAA ACC TTC GAT ACA GAA TAC CAA AAG
ACA AAG CTG AAC GAT TGG GAC TTT GTT GTG
ACA ACC GAC ATA TCT GAA ATG GGA GCC AAC
TTC AAG GCA GAC AGA GTG ATA GAT CCA AGG
AGA TGC TTG AAA CCT GTG ATA TTA ACA GAC
GGA CCT GAG AGA GTT ATC CTA GCT GGC CCA
ATG CCC GTG ACC GCC GCA TCA GCC GCT CAG
AGA AGG GGA AGG GTG GGA AGA AAC CCA CAA
AAA GAG AAT GAT CAA TAC ATC TTT ACC GGA
CAA CCT CTG AAC AAT GAC GAG GAC CAC GCT
CAT TGG ACA GAA GCA AAA ATG TTG CTC GAC
AAC ATT AAT ACT CCT GAG GGC ATT ATC CCC
GCC CTC TTT GAA CCT GAG AGA GAA AAG AGT
GCA GCT ATC GAC GGA GAG TAC AGG TTA AAA
GGA GAG TCA AGA AAA ACC TTC GTT GAA CTC
ATG AGG AGA GGC GAC TTA CCA GTC TGG TTG
GCA CAC AAA GTG GCC AGT GAA GGG ATA AAG
TAC ACA GAC AGA AAA TGG TGT TTC GAT GGA
CAA AGA AAC AAT CAG ATA CTC GAA GAG AAC
ATG GAC GTG GAA ATC TGG ACA AAA GAG GGA
GAA AAG AAA AAG CTA AGA CCA AGA TGG CTT
GAC GCT AGA ACA TAC TCA GAC CCT CTC GCC
CTT AAA GAA TTT AAA GAT TTT GCA GCT GGA
AGA AAA TCC ATA GCA CTC GAT CTG GTC ACA
GAG ATA GGG AGG GTC CCA TCA CAT TTG GCA
CAT AGG ACC AGA AAC GCT CTT GAT AAT CTG
GTG ATG CTA CAC ACC TCA GAA GAT GGA GGG
AGG GCA TAC AGG CAC GCC GTT GAG GAA TTA
CCC GAA ACA ATG GAA ACC CTC TTA CTG CTA
GGA TTA ATG ATA CTA CTG ACA GGC GGA GCA
ATG TTG TTT TTG ATT TCA GGG AAA GGA ATA
GGA AAA ACA AGC ATA GGG CTT ATA TGT GTG
ATA GCC TCA TCT GGA ATG CTT TGG ATG GCA
GAG GTG CCA CTA CAA TGG ATA GCC TCA GCA
ATC GTG CTT GAA TTT TTC ATG ATG GTG CTG
TTG ATA CCT GAA CCA GAA AAG CAA AGA ACA
CCA CAA GAC AAC CAA TTA GCA TAC GTT GTC
ATC GGA ATA TTG ACA TTA GCC GCA ACT ATA
GCC GCA AAC GAG ATG GGG CTA CTG GAG ACA
ACT AAA AGA GAT CTT GGG ATG TCC AAA GAA
CCA GGG GTT GTC TCC CCT ACA TCT TAC CTA
GAT GTG GAC CTA CAT CCT GCA TCT GCA TGG
ACA CTC TAT GCC GTT GCA ACC ACA GTG ATT
ACC CCA ATG TTA AGG CAT ACC ATC GAA AAC
TCT ACA GCC AAC GTG TCC CTA GCT GCA ATC
GCA AAC CAG GCC GTT GTG CTG ATG GGA TTA
GAT AAG GGC TGG CCA ATT AGT AAA ATG GAT
CTC GGA GTC CCC TTG CTA GCC CTG GGA TGC
TAT TCT CAG GTT AAT CCA TTA ACA TTA ACA
GCC GCT GTT CTC CTA CTT ATA ACC CAC TAC
GCA ATC ATA GGA CCA GGC CTG CAA GCC AAG
GCT ACA AGA GAA GCT CAA AAG AGA ACA GCA
GCT GGA ATT ATG AAA AAC CCA ACA GTG GAC
GGA ATC ATG ACA ATA GAT TTG GAT TCA GTC
ATT TTC GAT AGT AAA TTT GAG AAA CAG CTG
GGA CAA GTG ATG TTA CTC GTG CTT TGC GCT
GTT CAA CTT TTG CTA ATG AGA ACA TCC TGG
GCT TTG TGC GAG GCA TTA ACA TTG GCA ACT
GGA CCC ATA ACT ACA CTG TGG GAA GGA TCC
CCT GGG AAG TTC TGG AAC ACA ACC ATA GCA
GTG TCA ATG GCA AAC ATT TTT AGG GGA TCC
TAC TTG GCA GGA GCT GGA CTG GCC TTC TCC
ATT ATG AAA TCC GTT GGA ACC GGC AAA AGA
GGA ACC GGA TCA CAA GGA GAA ACA TTA GGA
GAA AAG TGG AAA AAG AAA TTG AAC CAA CTC
TCA AGA AAA GAA TTC GAT TTG TAT AAA AAG
TCA GGA ATT ACA GAA GTG GAC AGA ACA GAG
GCA AAG GAA GGC CTC AAG AGA GGA GAA ACA
ACT CAC CAT GCT GTC TCA AGG GGA TCA GCA
AAG TTA CAA TGG TTT GTC GAA AGA AAC ATG
GTG GTT CCA GAA GGC AGA GTG ATT GAC TTG
GGG TGT GGC AGA GGA GGG TGG TCA TAT TAC
TGC GCA GGG CTG AAA AAG GTG ACA GAG GTG
AGA GGA TAT ACA AAA GGA GGG CCA GGA CAT
GAA GAG CCA GTG CCA ATG TCC ACA TAC GGA
TGG AAC ATA GTC AAG TTA ATG AGT GGA AAA
GAC GTC TTC TAC CTA CCA CCC GAG AAA TGT
GAC ACC CTA CTT TGT GAT ATT GGC GAA TCA
TCT CCA TCT CCA ACA GTG GAA GAG AGC AGA
ACA ATT AGA GTG CTT AAA ATG GTG GAG CCA
TGG CTA AAA AAT AAC CAA TTT TGC ATT AAA
GTC CTT AAT CCA TAC ATG CCA ACA GTG ATC
GAA CAC CTG GAG AGG TTG CAG AGG AAA CAT
GGA GGC ATG CTT GTG AGA AAC CCA CTG TCA
AGA AAC AGC ACT CAT GAA ATG TAC TGG ATT
TCA AAC GGA ACA GGA AAC ATT GTT TCA AGC
GTT AAC ATG GTG TCC AGA CTG TTA CTT AAT
AGA TTT ACA ATG ACA CAC AGA AGG CCC ACA
ATT GAA AAA GAC GTG GAC CTA GGA GCC GGA
ACC AGA CAC GTG AAC GCA GAG CCC GAA ACA
CCT AAT ATG GAT GTG ATA GGG GAG AGG ATA
AAA AGA ATC AAA GAA GAG CAC AAC TCA ACA
TGG CAT TAT GAC GAT GAA AAC CCT TAT AAA
ACT TGG GCC TAC CAC GGA AGT TAT GAA GTT
AAG GCA ACA GGC AGT GCC TCT TCC ATG ATT
AAC GGA GTG GTT AAA TTG CTC ACT AAA CCT
TGG GAC GTG GTT CCA ATG GTT ACA CAA ATG
GCT ATG ACA GAC ACA ACC CCC TTT GGA CAA
CAG AGG GTC TTC AAG GAA AAG GTC GAC ACT
AGG ACC CCC AGA CCA ATG CCA GGA ACA AGG
AAA GCA ATG GAA ATA ACA GCA GAA TGG TTG
TGG AGA ACC CTG GGA AGA AAT AAA AGA CCA
AGG TTA TGC ACC AGA GAG GAA TTC ACA AAG
AAA GTG AGA ACA AAC GCA GCT ATG GGA GCA
GTG TTC ACA GAA GAG AAC CAA TGG GAC TCA
GCA AAA GCA GCC GTG GAA GAT GAA GAG TTT
TGG AAA CTC GTG GAT AGA GAA AGA GAA CTT
CAT AAA CTG GGA AAA TGT GGC TCA TGT GTG
TAC AAT ATG ATG GGA AAG AGA GAG AAG AAA
CTC GGA GAA TTC GGC AAG GCT AAA GGG TCA
AGG GCC ATT TGG TAT ATG TGG TTA GGG GCC
AGA TAC TTG GAA TTC GAG GCT CTA GGA TTT
CTT AAC GAA GAC CAC TGG TTC AGT AGG GAG
AAC TCA TAC TCA GGA GTG GAA GGA GAG GGA
CTG CAT AAA CTC GGA TAC ATT CTT AGA GAC
ATA AGT AAG ATA CCA GGA GGG GCC ATG TAC
GCA GAC GAT ACT GCA GGG TGG GAT ACA AGA
ATT ACA GAA GAC GAT CTG CAT AAC GAG GAA
AAA ATA ATC CAA CAG ATG GAC CCC GAA CAC
AGA CAA CTT GCT AAC GCC ATT TTT AAA CTA
ACT TAC CAA AAC AAG GTG GTC AAA GTC CAA
AGA CCA ACC CCT ACA GGA ACC GTT ATG GAC
ATA ATT TCC AGA AAG GAT CAG AGA GGC TCT
GGA CAA TTG GGA ACA TAC GGC TTG AAT ACC
TTT ACA AAC ATG GAA GCA CAA CTT GTG AGA
CAA ATG GAA GGG GAA GGG GTG CTG ACA AAA
GCT GAC TTG GAA AAC CCA CAT TTA CTA GAG
AAG AAA ATC ACA CAA TGG TTG GAA ACA AAG
GGA GTG GAG AGA CTG AAG AGA ATG GCT ATA
TCA GGC GAT GAC TGT GTG GTT AAG CCT ATA
GAC GAT AGG TTT GCC AAT GCA CTG CTC GCC
CTG AAT GAC ATG GGA AAG GTG AGA AAG GAT
ATT CCA CAA TGG CAA CCA TCA AAA GGG TGG
CAC GAT TGG CAA CAG GTG CCA TTT TGT AGC
CAC CAT TTC CAT GAG CTG ATA ATG AAA GAC
GGC AGA AAA TTA GTG GTC CCT TGT AGA CCA
CAA GAT GAG CTC ATA GGG AGA GCC AGA ATT
AGC CAG GGA GCC GGC TGG TCT CTG AGA GAG
ACT GCA TGT CTG GGA AAA GCC TAC GCA CAA
ATG TGG TCA CTG ATG TAT TTC CAC AGA AGG
GAC CTC AGA TTA GCA TCC AAT GCA ATA TGC
AGC GCA GTG CCA GTG CAC TGG GTG CCA ACA
TCA AGA ACA ACT TGG TCC ATA CAC GCC CAC
CAT CAA TGG ATG ACA ACA GAG GAC ATG CTG
ACA GTG TGG AAT AGA GTG TGG ATT GAA GAG
AAT CCA TGG ATG GAA GAT AAA ACA CCA GTC
ACA ACA TGG GAA AAC GTG CCA TAC TTG GGG
AAA AGA GAG GAT CAA TGG TGC GGA TCA CTC
ATA GGC TTG ACA TCC AGA GCA ACA TGG GCA
CAA AAC ATC CCA ACA GCC ATA CAG CAA GTG
AGA TCA CTT ATC GGG AAT GAG GAA TTT CTC
GAC TAC ATG CCC AGC ATG AAG AGA TTC AGA
AAG GAG GAA GAG TCT GAG GGG GCC ATT TGG
TAA
*GC% Content for gene (codon optimized): 60.06%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 3U1I_C GI:356624669
http://www.ncbi.nlm.nih.gov/protein/356624669
*Protein ID (NP or XP #) or Wolbachia#: Not found.
http://www.rcsb.org/pdb/explore/explore.do?structureId=3U1I
*Organism (including strain): Dengue Virus type 3 - Singapore/8120/1995
Viruses; ssRNA positive-strand viruses, no DNA stage; Flaviviridae; Flavivirus; Dengue virus group.
Etiologic Risk Group :
*Background/Disease Information :
Dengue Fever & Dengue Hemorrhagic Fever
( Also known as :Hemorrhagic dengue; Dengue shock syndrome; Philippine hemorrhagic fever; Thai hemorrhagic fever; Singapore hemorrhagic fever )
The four dengue viruses originated in monkeys and independently jumped to humans in Africa or Southeast Asia between 100 and 800 years ago. Dengue remained a relatively minor, geographically restricted disease until the middle of the 20th century.
The 4 dengue viruses (DENV) are immunologically related, positive-strand RNA viruses of the genus Flavivirus, family Flaviviridae. These viruses cause both dengue fever (DF) and dengue hemorrhagic fever (DHF). Asymptomatic infection also occurs. Infection with 1 DENV produces lifelong immunity against reinfection with that DENV type but no long-term cross-protection against the other 3 DENV types (any cross-protection is ≤2 months).
Early symptoms of dengue hemorrhagic fever are similar to those of dengue fever, but after several days the patient becomes irritable, restless, and sweaty. These symptoms are followed by a shock -like state.
Bleeding may appear as tiny spots of blood on the skin (petechiae) and larger patches of blood under the skin (ecchymoses). Minor injuries may cause bleeding.
Shock may cause death. If the patient survives, recovery begins after a one-day crisis period.
No specific therapeutic agents exist for dengue virus infections. Encourage bed rest and maintenance of fluids to prevent dehydration while the patient is febrile. Control fever with acetaminophen. Headache, eye pain, joint pain, and muscle ache may require narcotics. Aspirin, aspirin-containing drugs, and other nonsteroidal anti-inflammatory drugs (such as ibuprofen) should be avoided because of their anticoagulant properties. Aspirin and other salicylates should be especially avoided in children because of the association with Reye syndrome.
http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter-3-infectious-diseases-related-to-travel/dengue-fever-and-dengue-hemorrhagic-fever.htm
Essentiality of this protein: NS2B is a subunit in the serine protease enzyme that is essential for the DENV virus functioning.
Complex of proteins?: Forms a complex with the other subunit NS3p to form the NS2B(H)-NS3p complex.
Druggable Target:
http://www.sciencedirect.com/science/article/pii/S0006291X05005632
*EC#: 3.4.21.91 ( Specific for Dengue Virus 3)
Link to BRENDA EC# page:
http://brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.91&Suchword=&organism[]=Dengue+virus+3&show_tm=0
http://www.brenda-enzymes.org/index.php4?page=taxonomy2/ncbi/tree/index.php4?f[id][value]=11069
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://www.sciencedirect.com/science/article/pii/S0006291X05005632
http://www.sciencedirect.com/science/article/pii/S0166093403002556
-- link to Sigma (or other company) page for assay or assay reagents (substrates):
http://www.peptanova.de/products/Enzyme-Substrates/AMC-MCA-Substrates/Boc-Gly-Lys-Arg-AMC.html
-- paper that contains assay information:
http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&ved=0CDAQFjAA&url=http%3A%2F%2Fwww.peptanova.de%2Ffiles%2Famc3.pdf&ei=CHCCUYX9Cubx0gH9y4HADg&usg=AFQjCNHp8-s_1s0AX8fiMtFregQs6jyrqQ&bvm=bv.45921128,d.dmQ
-- List cost and quantity of substrate reagents and supplier
52,00 EUR*
Boc-Gly-Lys-Arg-AMC
Boc-GKR-AMC |Boc-Gly-Lys-Arg-MCA
3143-v 5 mg | 52.00 EUR
t-Butyloxycarbonyl- glycyl- L-lysyl- L-arginine- 4-methylcoumaryl- 7-amide
A trace of impurity might be detectable by TLC when 100 µg is applied to the plate. The amount of contamination detectable by HPLC is less than 2 %.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3U1I_C
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
http://www.uniprot.org/blast/uniprot/2013050226ZDIXWW3N
Current Inhibitors:
phthalazine-based compound, 7; pthalazine-based compound, 2; quanidine 5; quanidine 4; Bz-NKRR-H
Expression Information (has it been expressed in bacterial cells): Yes, in E-Coli
Purification Method:
http://www.jbc.org/content/275/14/9963.short
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
http://www.ncbi.nlm.nih.gov/protein/AFN80338.1
Considered sequence fragment (NS2B) : (1344 to 1473)
1321 1331 1341 1351 1361 1371
| | | | | |
1321 SWPLNEG VMAVGLVSIL ASSLLRNDVP MAGPLVAGGL 1380
1381 LIACYVITGT SADLTVEKAA DVTWEEEAEQ TGVSHNLMIT VDDDGTMRIK DDETENILTV 1440
1441 LLKTALLIVS GIFPYSIPAT LLVWHTWQKQ TQR
*length of your protein in Amino Acids:130 amino acids (from 1344 to 1473)
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 14065.2
http://web.expasy.org/cgi-bin/protparam/protparam1?Q5UB51@1344-1473@
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Didn't work..
*CDS Gene Sequence (paste as text only):
http://tinyurl.com/cnpgrrt
http://www.ncbi.nlm.nih.gov/nuccore/163644368?report=genbank&from=97&to=10269
1 mnnqrkktgk psinmlkrvr nrvstgsqla krfsrgllng qgpmklvmaf iaflrflaip
61 ptagvlarwg tfkksgaikv lkgfkkeisn mlsiinkrkk tslclmmmlp atlafhltsr
121 dgeprmivgk nergksllfk tasginmctl iamdlgemcd dtvtykcpli aevepedidc
181 wcnltstwvt ygtcnqageh rrdkrsvala phvgmgldtr tqtwmsaega wrqvekvetw
241 alrhpgftil alflahyigt sltqkvvifi llmlvtpsmt mrcvgvgnrd fveglsgatw
301 vdvvlehggc vttmaknkpt ldielqktea tqlatlrklc iegkitnitt dsrcptqgea
361 ilpeeqdqny vckhtyvdrg wgngcglfgk gslvtcakfq clepiegkvv qyenlkytvi
421 itvhtgdqhq vgndtqgvtv eitpqastve avlpeygtlg lecsprtgld fnemilltmk
481 nkawmvhrqw fldlplpwts gatterptwn rkellvtfkn ahakkqevvv lgsqegamht
541 altgateiqn sggtsifagh lkcrlkmdkl elkgmsyamc lntfvlkkev setqhgtili
601 kveykgedap ckipfstedg qgkahngrli tanpvvtkke epvnieaepp fgesnivigi
661 gdkalkinwy kkgssigkmf eatargarrm ailgdtawdf gsvggvlnsl gkmvhqifgs
721 aytalfsgvs wimkigigvl ltwiglnskn tsmsfscivi giitlylgav vqadmgcvin
781 wkgkelkcgs gifvtnevht wteqykfqad spkrlataia gawengvcgi rsttrmenll
841 wkqianelny ilwenniklt vvvgdtigvl eqgkrtltpq pmelkyswkt wgkakivtae
901 tqnssfiidg pntpecpsas rawnvweved ygfgvfstni wlklrevytq scdhrlmsaa
961 ikderavhad mgywiesqkn gswklekasf ievktciwpk shtlwsngvl esdmiipksl
1021 agpisqhnhr pgyhtqtagp whlgkleldf nycegttvvi tencgtrgps lrtttvsgkl
1081 ihewccrsct lpplrymged gcwygmeirp inekeenmvk slvsagsgkv dnftmgvlcl
1141 ailfeevmrg kfgkkhmiag vlftfvllls gqitwrdmah tlimigsnas drmgmgvtyl
1201 aliatfkiqp flalgfflrk ltsrenlllg vglamattlq lpedieqman gialglmtlk
1261 litqfetyql wtalvsltcs ntiftltvaw rtatlilagv sllplcqsss mrktdwlpmt
1321 vaamgvpplp lfifslkdtp krrswplneg vmavglvsil assllrndvp magplvaggl
1381 liacyvitgt sadltvekaa dvtweeeaeq tgvshnlmit vdddgtmrik ddeteniltv
1441 llktallivs gifpysipat llvwhtwqkq tqrsgvlwdv psppetqkae leegvyrikq
1501 qgifgktqvg vgvqkegvfh tmwhvtrgav lthngkrlep nwasvkkdli sygggwrlsa
1561 qwqkgeevqv iavepgknpk nfqtmpgifq tttgeigaia ldfkpgtsgs piinregkvv
1621 glygngvvtk nggyvsgiaq tnaepdgptp eleeemfkkr nltimdlhpg sgktrkylpa
1681 ivreaikrrl rtlilaptrv vaaemeealk glpiryqtta tksehtgrei vdlmchatft
1741 mrllspvrvp nynliimdea hftdpasiaa rgyistrvgm geaaaifmta tppgtaeafp
1801 qsnapiqdee rdiperswns gnewitdfvg ktvwfvpsik agndianclr kngkkviqls
1861 rktfdteyqk trlndwdfvv ttdisemgan fkadrvidpr rclkpviltd gpervilagp
1921 mpvtaasaaq rrgrvgrnpq kendqyiftg qplnndedha hwteakmlld nintpegiip
1981 alfepereks aaidgeyrlk gesrktfvel mrrgdlpvwl ahkvasegik ytdrkwcfdg
2041 ernnqileen mdveiwtkeg erkklrprwl dartysdpla lkefkdfaag rksialdlvt
2101 eigrvpshla hrtrnaldnl vmlhtsehgg rayrhaveel petmetllll glmilltgga
2161 mlflisgkgi gktsiglicv ivssgmlwma dvplqwiasa ivleffmmvl lipepekqrt
2221 pqdnqlayvv igiltlaaiv aanemgllet tkrdlgmske pgvvsptsyl dvdlhpasaw
2281 tlyavattvi tpmlrhtien stanvslaai anqavvlmgl dkgwpiskmd lgvpllalgc
2341 ysqvnpltlt aavlllvthy aiigpglqak atreaqkrta agimknptvd gimtidldpv
2401 iydskfekql gqvmllvlca vqlllmrtsw alcealtlat gpittlwegs pgkfwnttia
2461 vsmanifrgs ylagaglafs imksvgtgrr gtgsqgetlg ekwkkklnql srkefdlykk
2521 sgitevdrte akeglkrget thhavsrgsa klqwfvernm vipegrvidl gcgrggwsyy
2581 caglkkvtev rgytkggpgh eepvpmstyg wnivklmsgk dvfylppekc dtllcdiges
2641 spsptveesr tirvlkmvep wlknnqfcik vlnpymptvi ehlerlqrky ggmlvrnpls
2701 rnsthemywi sngtgnivss vnmvsrllln rftmthrrpt iekdvdlgag trhvnaepet
2761 pnmdvigeri krikeehsst whyddenpyk twayhgsyev katgsassmi ngvvklltkp
2821 wdvvpmvtqm amtdttpfgq qrvfkekvdt rtprpmpgtr kvmeitaewl wrtlgrnkkp
2881 rlctreeftk kvrtnaamga vfteenqwds araavedeef wklvdrerel hklgkcgscv
2941 ynmmgkrekk lgefgkakgs raiwymwlga rylefealgf lnedhwfsre nsysgvegeg
3001 lhklgyilrd iskipggamy addtagwdtr iteddlhnee kitqqmdpeh kqlanaifkl
3061 tyqnkvvkvq rptpkgtvmd iisrkdqrgs gqvgtyglnt ftnmeaqlvr qmegegvlsk
3121 adlenphlle kkitqwletk gverlkrmai sgddcvvkpi ddrfanalla lndmgkvrkd
3181 ipqwqpskgw hdwqqvpfcs hhfhelimkd grklvvpcrp qdeligrari sqgagwslke
3241 taclgkayaq mwslmyfhrr dlrlasnaic savpvhwvpt srttwsihah hqwmttedml
3301 tvwnrvwied npwmedktpv ttwedvpylg kredqwcgsl igltsratwa qniltaiqqv
3361 rsligdeefl dympsmkrfr keeesegaiw
*GC% Content for gene: 46.7 %
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATG AAC AAT CAA AGA AAA AAG ACA GGA AAG
CCC TCA ATC AAT ATG CTC AAA AGA GTC AGG
AAC AGG GTC AGC ACA GGA AGT CAA CTC GCT
AAA AGA TTC TCT AAA GGA CTG TTG AAC GGA
CAG GGA CCA ATG AAG TTG GTG ATG GCA TTC
ATC GCC TTT CTC AGA TTC CTC GCA ATA CCA
CCT ACA GCA GGG GTC TTA GCA AGA TGG GGA
ACA TTT AAA AAG AGT GGA GCT ATA AAG GTC
CTA AAA GGG TTT AAG AAA GAA ATA TCA AAT
ATG CTG TCC ATA ATT AAT CAG AGA AAA AAG
ACT AGT CTG TGT CTC ATG ATG ATA CTG CCA
GCT GCA CTG GCT TTC CAC CTC ACC TCA AGA
GAC GGA GAG CCA AGA ATG ATC GTG GGA AAA
AAC GAG AGA GGA AAG TCC CTT CTA TTT AAG
ACC GCC TCA GGA ATA AAC ATG TGT ACA CTC
ATC GCA ATG GAT CTT GGA GAA ATG TGC GAT
GAC ACA GTT ACA TAT AAG TGT CCA CAT ATC
ACA GAG GTT GAA CCA GAG GAT ATC GAC TGT
TGG TGT AAT CTC ACT TCA ACT TGG GTG ACT
TAC GGA ACA TGC AAC CAG GCA GGG GAG CAC
AGA AGG GAC AAA AGG AGC GTG GCT CTG GCA
CCA CAC GTG GGC ATG GGA CTC GAT ACC AGG
ACA CAG ACA TGG ATG TCT GCA GAG GGA GCT
TGG AGA CAA GTT GAA AAG GTT GAA ACA TGG
GCC TTG AGA CAT CCA GGA TTT ACC ATT TTG
GCC CTT TTT CTC GCA CAT TAT ATA GGA ACA
TCA CTT ACT CAA AAG GTG GTT ATC TTT ATA
CTC TTA ATG TTG GTG ACA CCA TCA ATG ACA
ATG AGG TGC GTT GGC GTG GGA AAC AGA GAC
TTC GTG GAG GGA CTT AGC GGA GCA ACC TGG
GTG GAT GTT GTC TTG GAA CAT GGC GGA TGC
GTC ACA ACT ATG GCA AAA AAC AAA CCA ACC
CTG GAT ATA GAA CTG CAA AAA ACT GAA GCA
ACT CAG TTG GCT ACA CTA AGA AAA CTA TGC
ATA GAA GGC AAA ATC ACT AAT ATT ACA ACC
GAC TCC AGG TGT CCA ACC CAA GGA GAG GCC
GTG TTA CCA GAG GAA CAG GAT CAA AAT TAT
GTG TGT AAA CAC ACT TAT GTG GAT AGA GGC
TGG GGA AAT GGA TGC GGG CTC TTC GGG AAG
GGC TCT CTC GTC ACA TGC GCA AAG TTC CAA
TGT TTG GAA CCA ATA GAA GGA AAG GTC GTG
CAA TAC GAG AAC CTT AAA TAT ACA GTT ATA
ATT ACA GTG CAC ACA GGA GAT CAA CAC CAG
GTG GGC AAT GAG ACA CAA GGA GTG ACA GCC
GAG ATA ACA CCC CAG GCT TCA ACA ACC GAA
GCA ATA TTG CCC GAA TAC GGA ACT CTG GGA
TTG GAG TGC AGC CCC AGA ACT GGA TTG GAT
TTT AAT GAA ATG ATA TTG CTG ACA ATG AAA
AAC AAG GCC TGG ATG GTC CAC AGA CAA TGG
TTT TTC GAC CTC CCA CTG CCA TGG GCA AGC
GGG GCT ACA ACT GAG ACC CCA ACT TGG AAT
AGG AAA GAA CTC CTT GTT ACA TTC AAA AAT
GCC CAC GCA AAA AAG CAA GAA GTT GTC GTG
CTT GGA AGC CAG GAA GGA GCT ATG CAC ACA
GCT CTC ACA GGG GCT ACA GAG ATA CAA AAC
TCA GGA GGC ACT TCC ATT TTT GCA GGC CAC
TTG AAG TGC AGA TTG AAA ATG GAT AAA CTG
GAA TTA AAG GGA ATG TCA TAC GCC ATG TGT
ACA AAT ACA TTC GTG CTG AAG AAA GAG GTT
AGC GAA ACA CAG CAT GGA ACC ATC TTA ATT
AAG GTG GAA TAT AAA GGA GAA GAC GCT CCT
TGT AAA ATT CCA TTT TCC ACT GAA GAC GGA
CAA GGA AAA GCA CAT AAC GGC AGA CTC ATC
ACC GCA AAT CCC GTG GTT ACA AAA AAG GAA
GAG CCT GTG AAC ATC GAA GCC GAG CCC CCA
TTC GGG GAG AGC AAT ATC GTG ATC GGA ATA
GGA GAC AAT GCA CTA AAA ATT AAT TGG TAT
AAA AAG GGA TCA AGC ATC GGA AAG ATG TTC
GAG GCT ACT GAA AGA GGA GCC AGA AGG ATG
GCC ATT TTG GGA GAT ACA GCA TGG GAC TTC
GGG TCC GTG GGA GGC GTC TTG AAT TCA CTG
GGA AAA ATG GTG CAC CAA ATA TTT GGA TCA
GCA TAT ACT GCA TTG TTC TCA GGA GTC TCA
TGG GTG ATG AAG ATA GGA ATA GGA GTC CTC
TTG ACA TGG ATT GGA CTG AAC TCA AAA AAT
ACA AGT ATG AGT TTC TCC TGT ATA GCT ATT
GGA ATA ATC ACC TTG TAC CTG GGA GCT GTT
GTG CAG GCA GAC ATG GGA TGC GTG ATT AAC
TGG AAA GGC AAA GAA CTG AAG TGC GGA TCT
GGC ATC TTT GTC ACA AAC GAG GTC CAT ACA
TGG ACA GAA CAG TAC AAG TTT CAA GCA GAT
TCA CCA AAA AGA CTC GCT ACT GCT ATT GCT
GGA GCA TGG GAG AAC GGA GTC TGT GGA ATA
AGA AGC ACC ACT AGA ATG GAA AAT TTG TTA
TGG AAG CAA ATA GCA AAT GAA CTG AAT TAT
ATA TTG TGG GAA AAT AAC ATA AAG TTA ACA
GTT GTG GTT GGA GAC ACA CTA GGA GTC CTA
GAA CAA GGA AAA AGG ACA CTG ACA CCT CAA
CCA ATG GAG TTA AAA TAC TCT TGG AAA ACA
TGG GGC AAG GCT AAG ATA GTG ACC GCA GAG
ACA CAG AAT TCA AGC TTC ATT ATA GAC GGC
CCA AAC ACT CCA GAA TGT CCA TCA GCC TCA
AGA GCT TGG AAT GTC TGG GAA GTC GAA GAT
TAC GGG TTC GGG GTC TTT ACC ACA AAT ATC
TGG CTG AAG CTG AGA GAA GTG TAC ACA CAG
TTG TGC GAC CAT AGA TTA ATG TCC GCA GCT
GTT AAG GAC GAG AGA GCA GTT CAC GCC GAC
ATG GGA TAT TGG ATT GAA TCA CAA AAA AAC
GGC TCT TGG AAA CTA GAG AAA GCA TCA CTC
ATC GAG GTG AAA ACT TGC ACA TGG CCA AAA
TCA CAC ACA TTG TGG ACA AAT GGA GTG TTA
GAG TCA GAT ATG ATA ATT CCA AAG TCC CTG
GCT GGG CCA ATT AGT CAA CAT AAC TAT AGA
CCA GGA TAT CAC ACA CAG ACA GCT GGG CCA
TGG CAC TTG GGA AAA CTT GAG TTG GAC TTT
AAT TAT TGT GAA GGG ACC ACT GTG GTT ATT
ACA GAG AGC TGC GGA ACA AGA GGA CCC TCC
CTA AGA ACC ACA ACT GTT TCT GGG AAA TTA
ATA CAC GAA TGG TGT TGC AGA TCA TGT ACA
TTG CCC CCA TTG AGG TAT ATG GGG GAG GAT
GGA TGT TGG TAT GGA ATG GAG ATA AGA CCA
ATA TCT GAA AAG GAG GAA AAC ATG GTT AAA
TCC CTT GTG AGC GCT GGA TCA GGG AAA GTT
GAC AAC TTC ACA ATG GGG GTG TTA TGC TTG
GCT ATT CTT TTT GAG GAA GTG CTA AGA GGA
AAG TTC GGA AAA AAG CAT ATG ATT GCC GGA
GTG TTC TTT ACA TTT GTC CTA TTA CTA TCA
GGG CAA ATA ACA TGG AGA GAC ATG GCA CAT
ACA CTA ATA ATG ATT GGA TCA AAC GCC TCA
GAC AGA ATG GGG ATG GGG GTT ACA TAC CTG
GCA CTG ATT GCA ACA TTT AAA ATT CAA CCA
TTC TTG GCC TTA GGG TTC TTT CTC AGA AAA
TTG ACA TCA AGG GAA AAC CTA TTA CTG GGA
GTC GGC CTG GCA ATG GCT ACA ACC CTA CAA
CTC CCA GAA GAC ATC GAA CAG ATG GCC AAC
GGG GTG GCA CTT GGC CTG ATG GCT CTT AAA
CTT ATA ACA CAA TTC GAA ACA TAT CAG CTT
TGG ACA GCT CTG GTT TCA CTT ACA TGT AGT
AAT ACA ATC TTC ACA CTT ACA GTG GCT TGG
AGA ACA GCC ACA TTG ATT CTA GCA GGA GTT
AGC CTT TTG CCC GTC TGC CAA TCA TCT AGC
ATG AGA AAG ACT GAT TGG TTG CCT ATG ACA
GTG GCT GCC ATG GGA GTG CCT CCA CTG CCT
CTT TTT ATT TTC TCA TTA AAA GAC ACA TTG
AAA AGA AGG TCA TGG CCA TTG AAC GAG GGG
GTT ATG GCA GTC GGA CTC GTG AGT ATC CTA
GCA TCA TCT TTA CTT AGA AAT GAC GTC CCA
ATG GCA GGA CCA TTG GTG GCC GGG GGA CTG
CTT ATT GCC TGC TAC GTC ATA ACA GGA ACT
TCA GCT GAT CTG ACT GTT GAA AAG GCA CCA
GAT GTG ACT TGG GAA GAG GAA GCC GAG CAA
ACA GGA GTC AGC CAT AAC CTA ATG ATA ACT
GTT GAC GAT GAC GGC ACA ATG AGA ATA AAG
GAT GAC GAA ACA GAA AAC ATA CTG ACA GTG
TTG CTG AAA ACA GCA TTG TTA ATC GTT TCT
GGC ATA TTC CCC TAT AGC ATT CCC GCC ACC
TTG TTA GTT TGG CAC ACA TGG CAA AAA CAA
ACC CAA AGA TCT GGA GTG CTG TGG GAC GTG
CCT TCA CCT CCC GAA ACA CAG AAA GCT GAG
CTA GAA GAG GGA GTC TAC AGA ATC AAA CAA
CAG GGG ATA TTT GGG AAA ACA CAA GTC GGC
GTG GGA GTG CAA AAG GAA GGC GTG TTT CAC
ACA ATG TGG CAC GTC ACA AGA GGG GCT GTC
TTG ACA CAT AAC GGA AAG AGA CTG GAG CCT
AAT TGG GCT AGT GTC AAG AAA GAT CTC ATC
AGC TAT GGA GGC GGG TGG AGG TTG TCA GCA
CAG TGG CAG AAG GGA GAG GAA GTG CAA GTG
ATA GCC GTG GAA CCC GGC AAA AAT CCA AAA
AAC TTT CAA ACC ACA CCC GGG ACC TTC CAA
ACA ACT ACA GGA GAA ATT GGG GCA ATA GCC
TTA GAC TTC AAA CCA GGG ACT TCT GGA TCA
CCC ATT ATC AAT AGG GAG GGA AAG GTG GTT
GGA CTG TAC GGC AAC GGA GTG GTT ACA AAG
AAC GGA GGG TAC GTG AGT GGA ATT GCA CAG
ACA AAT GCT GAA CCC GAT GGA CCA ACA CCA
GAA TTG GAG GAA GAG ATG TTT AAA AAG AGA
AAT TTG ACA ATA ATG GAT CTT CAC CCC GGA
TCA GGA AAG ACA AGG AAA TAC CTA CCA GCT
ATA GTG AGA GAA GCT ATA AAA AGG AGA TTG
AGG ACC TTG ATA CTA GCA CCA ACA AGA GTG
GTC GCT GCA GAG ATG GAG GAA GCA CTC AAA
GGA CTC CCT ATC AGG TAC CAG ACA ACT GCC
ACC AAA AGC GAA CAT ACT GGG AGA GAA ATC
GTC GAC TTG ATG TGC CAT GCC ACA TTC ACA
ATG AGA CTC CTA AGC CCC GTG AGG GTG CCA
AAC TAC AAC TTG ATT ATC ATG GAT GAG GCA
CAC TTT ACT GAC CCT GCC TCT ATT GCA GCT
AGA GGG TAT ATA AGC ACA AGA GTG GGG ATG
GGG GAA GCA GCC GCA ATA TTT ATG ACA GCC
ACA CCA CCT GGA ACT GCT GAT GCA TTC CCT
CAG TCA AAC GCC CCA ATA CAG GAT GAG GAA
AGA GAT ATC CCA GAA AGA TCA TGG AAC TCA
GGA AAT GAA TGG ATT ACA GAC TTC GCT GGC
AAG ACA GTG TGG TTT GTG CCA TCA ATT AAG
GCT GGC AAT GAC ATC GCC AAC TGT CTT AGA
AAG AAC GGA AAA AAG GTG ATA CAG CTA TCA
AGG AAA ACC TTC GAT ACA GAA TAC CAA AAG
ACA AAG CTG AAC GAT TGG GAC TTT GTT GTG
ACA ACC GAC ATA TCT GAA ATG GGA GCC AAC
TTC AAG GCA GAC AGA GTG ATA GAT CCA AGG
AGA TGC TTG AAA CCT GTG ATA TTA ACA GAC
GGA CCT GAG AGA GTT ATC CTA GCT GGC CCA
ATG CCC GTG ACC GCC GCA TCA GCC GCT CAG
AGA AGG GGA AGG GTG GGA AGA AAC CCA CAA
AAA GAG AAT GAT CAA TAC ATC TTT ACC GGA
CAA CCT CTG AAC AAT GAC GAG GAC CAC GCT
CAT TGG ACA GAA GCA AAA ATG TTG CTC GAC
AAC ATT AAT ACT CCT GAG GGC ATT ATC CCC
GCC CTC TTT GAA CCT GAG AGA GAA AAG AGT
GCA GCT ATC GAC GGA GAG TAC AGG TTA AAA
GGA GAG TCA AGA AAA ACC TTC GTT GAA CTC
ATG AGG AGA GGC GAC TTA CCA GTC TGG TTG
GCA CAC AAA GTG GCC AGT GAA GGG ATA AAG
TAC ACA GAC AGA AAA TGG TGT TTC GAT GGA
CAA AGA AAC AAT CAG ATA CTC GAA GAG AAC
ATG GAC GTG GAA ATC TGG ACA AAA GAG GGA
GAA AAG AAA AAG CTA AGA CCA AGA TGG CTT
GAC GCT AGA ACA TAC TCA GAC CCT CTC GCC
CTT AAA GAA TTT AAA GAT TTT GCA GCT GGA
AGA AAA TCC ATA GCA CTC GAT CTG GTC ACA
GAG ATA GGG AGG GTC CCA TCA CAT TTG GCA
CAT AGG ACC AGA AAC GCT CTT GAT AAT CTG
GTG ATG CTA CAC ACC TCA GAA GAT GGA GGG
AGG GCA TAC AGG CAC GCC GTT GAG GAA TTA
CCC GAA ACA ATG GAA ACC CTC TTA CTG CTA
GGA TTA ATG ATA CTA CTG ACA GGC GGA GCA
ATG TTG TTT TTG ATT TCA GGG AAA GGA ATA
GGA AAA ACA AGC ATA GGG CTT ATA TGT GTG
ATA GCC TCA TCT GGA ATG CTT TGG ATG GCA
GAG GTG CCA CTA CAA TGG ATA GCC TCA GCA
ATC GTG CTT GAA TTT TTC ATG ATG GTG CTG
TTG ATA CCT GAA CCA GAA AAG CAA AGA ACA
CCA CAA GAC AAC CAA TTA GCA TAC GTT GTC
ATC GGA ATA TTG ACA TTA GCC GCA ACT ATA
GCC GCA AAC GAG ATG GGG CTA CTG GAG ACA
ACT AAA AGA GAT CTT GGG ATG TCC AAA GAA
CCA GGG GTT GTC TCC CCT ACA TCT TAC CTA
GAT GTG GAC CTA CAT CCT GCA TCT GCA TGG
ACA CTC TAT GCC GTT GCA ACC ACA GTG ATT
ACC CCA ATG TTA AGG CAT ACC ATC GAA AAC
TCT ACA GCC AAC GTG TCC CTA GCT GCA ATC
GCA AAC CAG GCC GTT GTG CTG ATG GGA TTA
GAT AAG GGC TGG CCA ATT AGT AAA ATG GAT
CTC GGA GTC CCC TTG CTA GCC CTG GGA TGC
TAT TCT CAG GTT AAT CCA TTA ACA TTA ACA
GCC GCT GTT CTC CTA CTT ATA ACC CAC TAC
GCA ATC ATA GGA CCA GGC CTG CAA GCC AAG
GCT ACA AGA GAA GCT CAA AAG AGA ACA GCA
GCT GGA ATT ATG AAA AAC CCA ACA GTG GAC
GGA ATC ATG ACA ATA GAT TTG GAT TCA GTC
ATT TTC GAT AGT AAA TTT GAG AAA CAG CTG
GGA CAA GTG ATG TTA CTC GTG CTT TGC GCT
GTT CAA CTT TTG CTA ATG AGA ACA TCC TGG
GCT TTG TGC GAG GCA TTA ACA TTG GCA ACT
GGA CCC ATA ACT ACA CTG TGG GAA GGA TCC
CCT GGG AAG TTC TGG AAC ACA ACC ATA GCA
GTG TCA ATG GCA AAC ATT TTT AGG GGA TCC
TAC TTG GCA GGA GCT GGA CTG GCC TTC TCC
ATT ATG AAA TCC GTT GGA ACC GGC AAA AGA
GGA ACC GGA TCA CAA GGA GAA ACA TTA GGA
GAA AAG TGG AAA AAG AAA TTG AAC CAA CTC
TCA AGA AAA GAA TTC GAT TTG TAT AAA AAG
TCA GGA ATT ACA GAA GTG GAC AGA ACA GAG
GCA AAG GAA GGC CTC AAG AGA GGA GAA ACA
ACT CAC CAT GCT GTC TCA AGG GGA TCA GCA
AAG TTA CAA TGG TTT GTC GAA AGA AAC ATG
GTG GTT CCA GAA GGC AGA GTG ATT GAC TTG
GGG TGT GGC AGA GGA GGG TGG TCA TAT TAC
TGC GCA GGG CTG AAA AAG GTG ACA GAG GTG
AGA GGA TAT ACA AAA GGA GGG CCA GGA CAT
GAA GAG CCA GTG CCA ATG TCC ACA TAC GGA
TGG AAC ATA GTC AAG TTA ATG AGT GGA AAA
GAC GTC TTC TAC CTA CCA CCC GAG AAA TGT
GAC ACC CTA CTT TGT GAT ATT GGC GAA TCA
TCT CCA TCT CCA ACA GTG GAA GAG AGC AGA
ACA ATT AGA GTG CTT AAA ATG GTG GAG CCA
TGG CTA AAA AAT AAC CAA TTT TGC ATT AAA
GTC CTT AAT CCA TAC ATG CCA ACA GTG ATC
GAA CAC CTG GAG AGG TTG CAG AGG AAA CAT
GGA GGC ATG CTT GTG AGA AAC CCA CTG TCA
AGA AAC AGC ACT CAT GAA ATG TAC TGG ATT
TCA AAC GGA ACA GGA AAC ATT GTT TCA AGC
GTT AAC ATG GTG TCC AGA CTG TTA CTT AAT
AGA TTT ACA ATG ACA CAC AGA AGG CCC ACA
ATT GAA AAA GAC GTG GAC CTA GGA GCC GGA
ACC AGA CAC GTG AAC GCA GAG CCC GAA ACA
CCT AAT ATG GAT GTG ATA GGG GAG AGG ATA
AAA AGA ATC AAA GAA GAG CAC AAC TCA ACA
TGG CAT TAT GAC GAT GAA AAC CCT TAT AAA
ACT TGG GCC TAC CAC GGA AGT TAT GAA GTT
AAG GCA ACA GGC AGT GCC TCT TCC ATG ATT
AAC GGA GTG GTT AAA TTG CTC ACT AAA CCT
TGG GAC GTG GTT CCA ATG GTT ACA CAA ATG
GCT ATG ACA GAC ACA ACC CCC TTT GGA CAA
CAG AGG GTC TTC AAG GAA AAG GTC GAC ACT
AGG ACC CCC AGA CCA ATG CCA GGA ACA AGG
AAA GCA ATG GAA ATA ACA GCA GAA TGG TTG
TGG AGA ACC CTG GGA AGA AAT AAA AGA CCA
AGG TTA TGC ACC AGA GAG GAA TTC ACA AAG
AAA GTG AGA ACA AAC GCA GCT ATG GGA GCA
GTG TTC ACA GAA GAG AAC CAA TGG GAC TCA
GCA AAA GCA GCC GTG GAA GAT GAA GAG TTT
TGG AAA CTC GTG GAT AGA GAA AGA GAA CTT
CAT AAA CTG GGA AAA TGT GGC TCA TGT GTG
TAC AAT ATG ATG GGA AAG AGA GAG AAG AAA
CTC GGA GAA TTC GGC AAG GCT AAA GGG TCA
AGG GCC ATT TGG TAT ATG TGG TTA GGG GCC
AGA TAC TTG GAA TTC GAG GCT CTA GGA TTT
CTT AAC GAA GAC CAC TGG TTC AGT AGG GAG
AAC TCA TAC TCA GGA GTG GAA GGA GAG GGA
CTG CAT AAA CTC GGA TAC ATT CTT AGA GAC
ATA AGT AAG ATA CCA GGA GGG GCC ATG TAC
GCA GAC GAT ACT GCA GGG TGG GAT ACA AGA
ATT ACA GAA GAC GAT CTG CAT AAC GAG GAA
AAA ATA ATC CAA CAG ATG GAC CCC GAA CAC
AGA CAA CTT GCT AAC GCC ATT TTT AAA CTA
ACT TAC CAA AAC AAG GTG GTC AAA GTC CAA
AGA CCA ACC CCT ACA GGA ACC GTT ATG GAC
ATA ATT TCC AGA AAG GAT CAG AGA GGC TCT
GGA CAA TTG GGA ACA TAC GGC TTG AAT ACC
TTT ACA AAC ATG GAA GCA CAA CTT GTG AGA
CAA ATG GAA GGG GAA GGG GTG CTG ACA AAA
GCT GAC TTG GAA AAC CCA CAT TTA CTA GAG
AAG AAA ATC ACA CAA TGG TTG GAA ACA AAG
GGA GTG GAG AGA CTG AAG AGA ATG GCT ATA
TCA GGC GAT GAC TGT GTG GTT AAG CCT ATA
GAC GAT AGG TTT GCC AAT GCA CTG CTC GCC
CTG AAT GAC ATG GGA AAG GTG AGA AAG GAT
ATT CCA CAA TGG CAA CCA TCA AAA GGG TGG
CAC GAT TGG CAA CAG GTG CCA TTT TGT AGC
CAC CAT TTC CAT GAG CTG ATA ATG AAA GAC
GGC AGA AAA TTA GTG GTC CCT TGT AGA CCA
CAA GAT GAG CTC ATA GGG AGA GCC AGA ATT
AGC CAG GGA GCC GGC TGG TCT CTG AGA GAG
ACT GCA TGT CTG GGA AAA GCC TAC GCA CAA
ATG TGG TCA CTG ATG TAT TTC CAC AGA AGG
GAC CTC AGA TTA GCA TCC AAT GCA ATA TGC
AGC GCA GTG CCA GTG CAC TGG GTG CCA ACA
TCA AGA ACA ACT TGG TCC ATA CAC GCC CAC
CAT CAA TGG ATG ACA ACA GAG GAC ATG CTG
ACA GTG TGG AAT AGA GTG TGG ATT GAA GAG
AAT CCA TGG ATG GAA GAT AAA ACA CCA GTC
ACA ACA TGG GAA AAC GTG CCA TAC TTG GGG
AAA AGA GAG GAT CAA TGG TGC GGA TCA CTC
ATA GGC TTG ACA TCC AGA GCA ACA TGG GCA
CAA AAC ATC CCA ACA GCC ATA CAG CAA GTG
AGA TCA CTT ATC GGG AAT GAG GAA TTT CTC
GAC TAC ATG CCC AGC ATG AAG AGA TTC AGA
AAG GAG GAA GAG TCT GAG GGG GCC ATT TGG
TAA
*GC% Content for gene (codon optimized): 60.06%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
AssayMethodsUsingPeptidyl-AMC(MCA)Substrates(3)
(ExampleforCaspase-7usinganAuto-FluorescenceSpectrophotometerforMultiplate)
Reagents
1.Substratestocksolution:Contentofvial(Code3171-v Ac-DEVD-AMC), inDMSOat10mM
2.AMC stocksolution:Contentofvial(Code3099-vAMC), inDMSOat1mM
3.Buffer:ICEstandardbuffer(100mMHEPES-KOH,pH7.5, 10%sucrose(w/v),
0.1%CHAPS(w/v),10mMDTT, 0.1mg/mlovalbumin)
4.Enzymesolution:rec. Caspase-7inbuffer
Procedure
a)Initialrateassayfor30minatλex=390nm, λem=460nm
b)End-point(30min)assayat5different substrateconcentrationsatλex=355nmor380nm,λem=460nm
1.
2.Substrate:Dilutethestocksolutionwiththebuffer (x10, x20,x40, x80, x160)
3.Caspase-7:Dissolveinbuffer
4.Pipette160 µlofbufferand20Mlofsubstrate solutions(1, ½,¼, 1/8, 1/16mM)ineachwell
5.Incubateinthefluorescencespectrophotometer for3-4min(fortemperatureequilibration)
6.Takethemultiplateoutand add20 µlofenzymesolutionineachwell
7.Mounttheplateinthefluorescencespectrometer
8CalculatetheamountofreleasedAMC
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